How proteins are changed to recombinant proteins? Since proteins are involved in virtually all cellular processes, they are key subjects of interest for laboratory researchers in life sciences (e. g. in high-throughput assays such as ELISA, …) as well as physicians in the clinic.
In a way, recombinant proteins are tailor-made, engineered proteins to either mimic their natural counterparts or have specialized properties to be used as research reagents (e. g. high-affinity fluorescence probes) or biopharmaceuticals (most prominently in oncology).
In order to provide an answer to the initial question, we will discuss what constitutes recombinant proteins and how they differ from normal proteins. Additionally, we will give an overview of the necessary production steps, a brief history of recombinant protein technology and highlight examples of types of recombinant proteins.
In general, proteins are long chains of connected amino acids that fold into particular 3D structures and carry out functions in cells, depending on the sequence of amino acids. Their functions range from accurate folding of other proteins (e. g. chaperones) to metabolic catalysis (enzymes like proteases) to regulatory pathways (hormones, receptors) and immunity (antibodies, immunoglobulins).
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Their sequence information is stored in genes (DNA), which can be considered genetic blueprints of the respective proteins. DNA is the template for messenger RNA synthesis (transcription) which contains regulatory elements in introns, while exons carry the protein sequence. MRNA acts as the template during protein expression in ribosomes. Higher organisms are capable of altering primary expression products by post-translational modifications, e. g. disulfide bonds, phosphorylation, glycosylation.
Natural proteins need to be isolated ex vivo from cells or tissues and the efficiency of the process depends on the naturally occurring concentration and distributions.
On the other hand, recombinant proteins are artificially made, using molecular biology techniques and cell cultures. The biggest advantages are the potential to produce large amounts without the need for sufficient natural sources and the possibility of engineering the sequence to fine-tune their properties or create completely novel proteins. Therefore, a recombinant protein from the biotechnology factory may be indistinguishable from a natural protein of the same sequence from a tissue sample, but their production is entirely different.
Generally, modifications are done at the genetic level, using recombinant technology. To obtain relevant DNA sequences, mRNAs (hence exons only) are isolated and reverse-transcribed into cDNA. The genes of interest are flanked with regulatory elements to enhance polymerase recruitment and facilitate high yields (promoters). The resulting recombinant vectors are introduced into cell cultures, which in turn begin to produce the desired proteins.
Read more in the article: What is recombinant?
Production of recombinant proteins on larger scale can be considered a high-tech process. A fully equipped biotech laboratory and skilled labor are needed for high quality products.
Moreover, several components are required in the process:
Currently, the most popular expression systems are probably E. coli for polypeptides and simple proteins, due to their long-standing cell biological characterization and good yield, and Chinese Hamster Ovary cells (CHO cells) for human proteins due to their reliability, highest quality and great yield.
The expression of recombinant proteins (heterologous protein expression) involves a lot of key steps and specificities, depending on the type of target protein, e. g. sequence generation for antibodies (phage display, single B cell technologies) and peptide hormones (gene synthesis).
These general steps are common for all recombinant proteins:
In the next section, we will take a closer look into the different phases of recombinant protein production.
First of all: What is recombinant DNA? Generally speaking, recombinant DNA is made with molecular biology techniques, using natural sources of genetic material possibly from different species, combining them in novel ways (hence “recombinant”; so-called fusion proteins).
Another possibility is completely artificial, in vitro made recombinant DNA, using the technique of gene synthesis with overlapping single stranded DNA fragments, heating, annealing, ligation and amplification with PCR. This gives total control of the codon sequence and terminator, and allows site-specific mutation of residues to create derivates, thus generating novel variants of wild-type sequences.
The sequence coding for the proteins as is will generally be flanked with special codes to enhance replication and protein expression as well as a resistance gene for antibiotics to form a vector, which will be used in the next step.
The recombinant DNA vector needs to be introduced into the host cells (so-called protein expression systems) in processes termed transformation (for simpler cells and plant cells) and transfection (for animal cells). This can be achieved by chemical, mechanical and electric means.
Remember the antibiotic resistance gene in the vector? It is used to selectively kill cells that did not take up the vector by adding the antibiotic, which will do no harm to transformed/transfected cells.
Those cells will be reprogrammed to express the protein of interest while they grow, sometimes enriching the recombinant protein in inclusion bodies. Mammalian cells will even take care of proper protein folding and post-translational modifications like site-specific disulfide bonds, phosphorylation, and glycosylation.
After the cell growth is at the end of the exponential stage, the cell culture will be terminated, and the downstream processing is commenced.
Downstream processing starts with opening of the cells to release their contents: the protein of interest and all other cellular constituents. This process is termed cell lysis and is effected by three classes of mechanisms:
The choice of lysis method depends on the stability of the protein to ensure it keeps its native conformation. Since the cells contain proteases that digest proteins, sensitive products must be protected by addition of protease inhibitors.
Subsequently, the protein of interest must be separated from cell debris and unwanted cellular constituents (nucleic acids, fats, salts, etc.). Next, the protein fraction is subjected to protein purification methods such as fractional precipitation, size exclusion chromatography, Tag-affinity separation (using proteins with tags on either N-terminal or C-terminal end), ion-exchange chromatography to isolate the desired protein product.
The history of recombinant protein production began in the 1970s in the USA, when the first publications describing the enzymatic joining of DNA molecules were published1 and spawned an exciting series of milestone discoveries and development concerning the production of recombinant proteins:
Read more about the history of antibodies.
Nowadays, there are many types of recombinant proteins, ranging from reagents for diagnostic assays, molecular tools for life science researchers, enzymes for industrial applications (e. g. feedstock chemicals conversion, cleaning agents), but the most significant impact to mankind is probably in the class of therapeutic proteins:
The development of each of these types of proteins has had an enormous impact on patients, either saving their lives or improving their conditions.
1.Lobban PE, Kaiser AD. Enzymatic end-to-end joining of DNA molecules. Journal of Molecular Biology. Published online August 1973:453-471. doi:10.1016/0022-2836(73)90468-3
2.Jackson DA, Symons RH, Berg P. Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli. Proc Natl Acad Sci USA. Published online October 1972:2904-2909. doi:10.1073/pnas.69.10.2904
3.Itakura K, Hirose T, Crea R, et al. Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin. Science. Published online December 9, 1977:1056-1063. doi:10.1126/science.412251
4.The Nobel Prize for Chemistry 1980. The Nobel Prize. Accessed December 2022. https://www.nobelprize.org/prizes/chemistry/1980/summary/
5.McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. Published online December 1990:552-554. doi:10.1038/348552a0
6.Mori K, Kuni-Kamochi R, Yamane-Ohnuki N, et al. Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnol Bioeng. Published online 2004:901-908. doi:10.1002/bit.20326